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Abstract:

An assay for assessing the risk of disease in an individual, wherein said
assay comprises the steps of isolating a population of cells from normal
tissue of said individual, and quantitatively determining the frequency
of epimutation of a particular gene in said population of cells, wherein
the epimutation of said gene is associated with said disease and said
gene is other than one that is subject to normal parent of
origin-specific expression. Preferably, the epimutation is DNA
methylation of a tumour suppressor gene such as hMLH1, hMSH2, APC 1A, APC
1B and p16.

Claims:

1. An assay for assessing the risk of disease in an individual, wherein
said assay comprises the steps of;(i) isolating a population of cells
from normal tissue of said individual, and(ii) quantitatively determining
the frequency of epimutation of a particular gene in said population of
cells, wherein the epimutation of said gene is associated with said
disease and said gene is other than one that is subject to normal parent
of origin-specific expression.

2. The assay of claim 1, wherein the normal tissue is selected from the
group consisting of normal peripheral blood, normal hair follicle tissue
and normal tissue from the buccal cavity.

5. The assay of claim 1, wherein the epimutation is present in the
promoter or other regulatory region of the gene and is associated with
transcriptional silencing of said gene.

6. The assay of claim 1, wherein the epimutation is associated with
cancer.

7. The assay of claim 1, wherein the epimutation is present in a tumour
suppressor gene.

8. The assay of claim 7, wherein the epimutation is present in a gene
selected from the group consisting of hMLH1, hMSH2, APC1A, APC1B and p16.

9. The assay of claim 8, wherein the epimutation is present in hMLH1.

Description:

FIELD OF THE INVENTION

[0001]The present invention relates to an assay for assessing the risk of
disease (e.g. cancer) in an individual. In particular, the present
invention relates to an assay for assessing the risk of disease
comprising quantitatively determining the frequency of an epimutation in
a particular gene in a population of cells from normal tissue of an
individual, wherein epimutation of the gene is associated with one or
more diseases.

BACKGROUND OF THE INVENTION

Epigenetic Modifications and Gene Expression

[0002]Epigenetic modifications are molecular events that result in
alterations in gene function that are mediated by factors other than a
change in DNA sequence. Epigenetic effects on gene function commonly
result in transcriptional silencing of the gene that may be maintained
through mitosis, producing clonal patterns of transcriptional silence.
Silencing may occur with a probability that is somewhere between 0 and 1,
producing, in a single multicellular organism, a mosaic pattern of gene
expression (or silence). This mosaic expression occurs despite all cells
having the same genetic makeup. In some cases, epigenetic modifications
are maintained in the germ-line, producing heritable effects ("epigenetic
inheritance"). The molecular basis of epigenetic effects is much more
complex than the simple 4-base code in DNA, and for this reason,
epigenetic inheritance occurs in patterns that are much different from
the simple patterns of Mendelian inheritance.

[0003]One of the best known epigenetic modifications is cytosine
methylation ("DNA methylation"), which is indispensable for normal human
development and is involved in the normal physiological processes of
parental imprinting, suppression of transposable elements, and
X-inactivation in females (reviewed in Jones and Takai 2001, and Bird
2002). In all mammals, cytosine methylation occurs essentially within the
dinucleotide CpG. In the human genome, the majority of cytosine residues
within CpG dinucleotides are methylated, but small proportions are
maintained as unmethylated in certain CpG-rich regions called "CpG
islands" (Antequera and Bird 1993). CpG islands are frequently associated
with the regulatory regions of cellular genes, and a large proportion of
human genes include a CpG island at their 5' end.

[0004]Histone and chromatin structure changes are other epigenetic
modifications which affect gene expression. Indeed, both of these kinds
of epigenetic modifications have been found to have a great impact on
gene expression that is linked, although not exclusive to, DNA
methylation within CpG islands (Jenuwein and Allis 2001). For example,
transcriptionally active genes are generally associated with the
acetylation of the fourth lysine (K4) of the histone subunit 3
(H3K4), whereas silent and methylated genes are correlated with
de-acetylated H3K4, methylation of H3K9, and recruitment of the
HP1 chromodomain (Kouzarides 2002). In fact, recent evidence indicates
that DNA methylation occurs in response to a change in chromatin
structure that is largely dictated by modifications to these key histone
subunits (Tamaru and Selker 2001), and indicates that the role of DNA
methylation is a consolidation of the already silent state. Therefore,
DNA methylation can be regarded as a "signature" of a stably silenced
genetic locus.

[0005]DNA methylation is not, however, an absolute requirement or
"signature" for gene silencing since many non-human species which are
devoid of CpG methylation still exhibit epigenetic silencing phenomena.
Therefore, in the human genome, there are presumably many genes devoid of
CpG island promoters that will still be susceptible to epigenetic
modification mediated by changes in histones and chromatin structure,
rather than DNA methylation. The ease and low cost with which DNA
methylation can be assayed, however, makes it an attractive target to
search for epigenetic modifications in humans.

Methods to Detect Epigenetic Changes

[0006]As described above, the simplest way to determine an epigenetic
change is to test for CpG methylation. Traditionally, CpG methylation
analysis has been carried out by Southern hybridisation, which assesses
methylation-sensitive restriction enzyme sites within CpG islands of
known genes, however, recently, more sophisticated methods for
determining CpG methylation such as COBRA (combined bisulfite restriction
analysis; Xiong and Laird 1997), bisulfite allelic sequencing (Frommer et
al 1992), and MSP (methylation-specific PCR), have become available and
allowed a more detailed analysis of CpG methylation across a CpG island
of interest.

[0007]In particular, bisulfite modification of DNA now allows the
discrimination of methylated CpG from unmethylated CpG, since the
bisulfite treatment converts unmethylated cytosine to uracil through
deamination whereas 5-methylcytosine is protected from deamination and
thereby remains unchanged. Following treatment with bisulfite, the method
requires that the bisulfite-modified sequence be amplified by PCR with
strand-specific primers to yield a product in which uracil residues are
amplified as thymine, and only 5-methylcytosine residues are amplified as
cytosine. The PCR products can then be readily digested with restriction
enzymes to distinguish methylated from unmethylated alleles (COBRA), or
cloned and sequenced to provide methylation maps of individual DNA
strands.

[0008]MSP, another widely used methylation assay method, can assess the
methylation status of CpG sites within a CpG island, independent of the
use of methylation-sensitive restriction enzymes. In this method,
bisulfite modification is followed by amplification with primers specific
for methylated DNA only, and results in the amplification of any
hypermethylated alleles within a given sequence (U.S. Pat. No.
5,786,146).

[0009]Further, methods for detecting epigenetic modifications are not
limited to analysis of CpG methylation. That is, epigenetic modifications
can also be detected by various methods which assay specific proteins
bound to transcriptionally active or silent regions of DNA, or protein
modifications associated with active or silent states (e.g. detection of
specific modifications of histones, and the detection of other proteins
such as homologues of HP1). At present, these protein modifications are
generally assayed by immunoprecipitation with antibodies and subsequent
analysis for DNA sequences present in the precipitated material.

Genetic Basis of Disease

[0010]Efforts to predict predisposition for a disease, such as cancer,
have been to date based largely on age, personal or family history, or
occasionally by the inheritance of genetic abnormalities (i.e. germ-line
mutations). For example, mutations in the BRCA genes are present in
around half of all individuals with a strong family history of breast
cancers. However, this accounts for less than 1% of breast cancers
overall, and in the great majority of remaining cases there is no
familial pattern consistent with a defect in a single gene. A similar
situation exists with a large number of other diseases. Indeed, Table 1
provides a list of over sixty diseases which have been linked to certain
genes, but where only a relatively small proportion of cases can be
explained by a single predisposing genetic change.

[0011]A common explanation for diseases that have some familial pattern
indicating inheritance, but no evidence for a single predisposing genetic
change, is that disease results from the interaction of multiple genes.
In line with this, a trait (which can manifest as disease) may be
produced by the combined action of several genes, but only certain
alleles of those genes will contribute to the trait. Such traits have
been termed "quantitative traits", "complex traits" and "polygenic
traits", and diseases that may result from such a mechanism are typically
known as "multifactorial" or "polygenic" (for reviews, see Risch 2000,
and Botstein and Risch 2003).

[0012]Genetic studies can not therefore, predict predisposition to disease
in most cases. However, the fact that only a few patients in a family
showing a strong history of a disease such as breast cancer are likely to
show a single predisposing genetic change, suggests that many individuals
have an innate predisposition to cancer, the basis of which is currently
unknown.

Epimutations

[0013]A gene may be inactivated by epigenetic modification. The term
"epimutation" was first defined by Holliday (Holliday 1987) as a
"mitotically heritable change in the methylation of a gene", however the
term has since been extended to refer also to the other types of
epigenetic modifications. As used herein, the term "epimutation" refers
to any abnormal silencing of gene expression, in the absence of DNA
sequence alteration. This definition specifically excludes abnormal
silencing of a gene that is normally subject to parental imprinting (also
termed "genomic imprinting" or simply "imprinting"). Parental imprinting
is a normal process that involves changes in the transcription state of
one allele of a gene determined by the parental origin of the allele
(i.e. a change in the transcription state of one allele of a gene that is
normally subject to parent of origin-specific expression). This process
sometimes is aberrant, resulting in the loss of monoallelic expression
and thus expression that is either biallelic or completely absent.

PRIOR ART

[0014]To date, the only clear case of a germ-line epimutation comes from a
naturally occurring variant of the flowering plant, toadflax (Linaria
vulgaris) (Cubas et al 1999). In this example, biallelic methylation and
transcriptional silencing of a gene controlling symmetry, Lcyc, was found
to be the cause of an alteration in flower phenotype. The phenotype was
found to be somewhat unstable with a tendency to revert (i.e. some
flowers on mutant plants exhibited a wild type appearance and this was
associated with a loss of Lcyc methylation). Plants exhibiting the mutant
phenotype were able to transmit the epimutation to their progeny through
the germ-line. Germ-line and soma are not well separated in plants
however, and this may explain the relatively stable existence of this
epimutation over at least 250 years.

[0015]Epimutations are common in tumour cells. There is now a large body
of literature documenting epimutations in many types of tumour, and their
inverse relationship to activity of the affected gene (for reviews, see
Jones and Laird 1999, Wolffe and Matzke 1999, and Baylin and Herman
2000). In some cases, the epigenetic silencing of tumour suppressor genes
gives rise to distinct tumour phenotypes. For example, in sporadic
colorectal cancer, around 15% of tumours exhibit microsatellite
instability (MSI). MSI is a hallmark of defective mismatch repair, but
only a tiny fraction of these cancers will be explained by a genetic
alteration in a mismatch repair gene. It is now known that bi-allelic
methylation of the hMLH1 gene promoter is responsible for MSI tumours in
the majority of cases (Herman et al 1998, and Wheeler et al 1999). MSI
colorectal cancers also exhibit loss of imprinting (LOI) at IGF2 (Cui et
al 1998), which may also have an epigenetic basis (Cui et al 2002). It
has also been demonstrated that LOI could be detected not only in MSI
tumours, but also in the normal tissues of such patients, including their
peripheral blood (Cui et al 2003). The finding of LOI in peripheral blood
in a small number of normal controls, and a large percentage of
colorectal cancer patients has led to the hypothesis that LOI in
peripheral blood is an indicator of colorectal cancer risk (U.S. Pat. No.
6,235,474).

[0016]Germ-line epimutations have not yet been described in humans,
although a related phenomenon can be observed in a particular strain of
inbred mice, the agouti viable yellow (Avy). These mice carry the
Avy allele, in which an intracisternal A particle (IAP)
retrotransposon is inserted at the 5' end of the agouti (A) gene (Duhl et
al 1994). When the IAP is epigenetically active, agouti transcription is
initiated from a cryptic promoter within the 5' LTR of the IAP. The tight
tissue-specific expression of agouti is abrogated by the IAP, whose LTR
is active in many or all tissues and, as a result, agouti may be
expressed pancellularly in Avy mice. It has been found that the CpG
methylation of this IAP is inversely correlated with ectopic agouti
expression, and this epigenetic modification appears to gives rise to a
variation in phenotype in Avy mice which includes not only yellow
coat colour, but also obesity, Type II diabetes, and tumour
susceptibility. Significantly, this phenotype is mosaic in many
individuals, indicating that the IAP is active in some cells, and silent
in others, in a clonal pattern. Further, recent data indicates that there
is incomplete erasure of the Avy IAP methylation between generations
resulting in partial maternal inheritance of the epigenotype (Morgan et
al 1999). This suggests that germ-line transmission of an epigenetic
modification is possible in mammals.

Need for Methods for Assessing Disease Risk

[0017]New and improved methods for assessing disease risk in individuals
(i.e. predicting predisposition for a disease) are desirable. That is,
knowledge of disease risk may allow for the adoption of preventative
therapies and avoidance of disease risk factors, and may further assist
in the identification of preferred therapies upon commencement of the
disease or symptoms. Preferably, methods for assessing risk are
relatively simple and either non-invasive or cause only minimal
discomfort to individuals.

[0018]The present applicants have detected epimutation (i.e. CpG
methylation) in the promoter of the tumour suppressor gene hMLH1, in
normal tissues (e.g. peripheral blood) of cancer patients with tumours
showing a loss of the hMLH1 protein, and have found, surprisingly, that
the frequency of the detected epimutation in the cells of such normal
tissues is predictive of the level of cancer risk.

SUMMARY OF THE INVENTION

[0019]Thus, the present invention provides an assay for assessing the risk
of disease in an individual, wherein said assay comprises the steps of;

(i) isolating a population of cells from normal tissue of said individual,
and(ii) quantitatively determining the frequency of epimutation of a
particular gene in said population of cells, wherein epimutation of said
gene is associated with said disease and said gene is other than one that
is subject to normal parent of origin-specific expression.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 shows hMLH1 COBRA methylation analysis in peripheral blood in
Example 1. (A) This photograph shows an example of the COBRA screening
assay. In particular, results are shown from the C region COBRA in
peripheral blood DNA from 44 cancer patients. In this subset, one patient
showed methylation of hMLH1 in the C region demonstrated by digestion of
the PCR product (upper band) to yield two smaller fragments, which appear
as one band (arrow). (B) These photographs show the A, B and C region
COBRA results for the peripheral blood of individuals VT and TT. The
location of each region relative to the transcription start site is shown
on the left. For each region, digestion of the PCR product (upper band)
to yield smaller fragments is indicative of methylation within that
region. +, RKO cell line; -, healthy control blood DNA.

[0021]FIG. 2 shows immunohistochemical analysis of hMLH1 expression in
representative cancers from the two individuals in Example 1. Carcinomas
in the upper panel are from VT (left=breast, middle=endometrium,
right=colon) and those in the lower panel are from TT (left=colon,
middle=ampulla of Vater. Right=duodenal). All cancers showed complete
loss of hMLH1 expression. For all tumours, the inset shows positive
staining of the same tumour for hMSH2. Immunoperoxidase with haematoxylin
counterstain; Bar (lower left) represents 100 μm.

[0022]FIG. 3 shows bisulfite sequencing analysis of VT and TT somatic
tissues in Example 1. (A) Schematic representation of the hMLH1 locus
showing the locations of the A, B, and C regions in relation to the
region sequenced (dotted lines). (B) Sequence of hMLH1 within the dotted
region defined in (A). The primers used to amplify this region are
underlined. CpG doublets within this domain are highlighted in bold and
numbered 1 through 17. The single nucleotide polymorphism is also
highlighted with G and A shown in larger text. (C) This figure shows the
results of bisulfite allelic sequencing in the various somatic tissues of
TT and VT. Black and white squares represent individual CpGs and are
numbered according to their location in the sequence shown in (B). Grey
or white circles represent the A or G genotype, respectively. Each
horizontal row of squares represents the results from individual alleles.
In both patient TT and VT, the hypermethylated alleles are always of the
G genotype, whereas the A alleles show patchy methylation only and are
never hypermethylated. Mosaicism was evident in the hair follicles of TT,
and in all tissues from VT, as evidenced by the hypomethylation of the
occasional G allele.

[0023]FIG. 4 shows the results of bisulfite sequencing analysis of TT
sperm in Example 1. (A) This photograph shows the results of the hybrid
MSP-COBRA PCR used to amplify methylated alleles from the purified sperm
from patient TT. Note the weak amplification of sperm compared to the
positive control cell line (+). No amplification was seen in the negative
control (peripheral blood from a healthy donor). (B) This figure shows
the results of bisulfite allelic sequencing of the fragment from sperm
shown in (A). Black and white squares represent individual CpGs and are
numbered according to their location in the sequence shown in FIG. 3(B).
Grey or white circles represent the A or G genotype, respectively. Each
horizontal row of squares represents the results from individual alleles.
In the sperm, only G alleles were hypermethylated whereas the A alleles
are hypomethylated. Mosaicism was evident with 10 of 16 G alleles
demonstrating hypomethylation.

[0024]FIG. 5 provides the results of analysis of hMLH1 methylation in
normal bowel tissue from cancer patients in Example 1. (A) This
photograph shows an example of the COBRA screening assay in the normal
bowel tissue from 14 cancer patients. Shown are the results from the
hMLH1 C region COBRA. In this subset, one patient showed methylation of
hMLH1 in the C region in normal bowel tissue, demonstrated by digestion
of the PCR product (upper band) to yield two smaller fragments, which
appear as one band (arrow). (B) This figure shows the results of
bisulfite allelic sequencing of the fragment from the normal bowel shown
in (A). Black and white squares represent individual CpGs and are
numbered according to their location in the sequence shown in FIG. 3B.
White circles represent the G genotype. Each horizontal row of squares
represents the results from individual alleles. Hypermethylated alleles
were clearly present and were always of the G genotype. This patient is a
GG homozygote for the hMLH1 SNP thus mosaicism cannot be determined.

[0025]FIG. 6 provides representative bisulfite sequencing of MSP products
from healthy individuals assayed in Example 2. Each horizontal row of
squares represents the results from individual alleles. Black and white
squares represent individual CpGs that are either methylated, or
unmethylated, respectively. Hypermethylated alleles were clearly present
in healthy individuals in both the hMLH1 (A) and p16%) genes.

DETAILED DESCRIPTION OF THE INVENTION

[0026]The present invention provides an assay for assessing the risk of
disease in an individual, wherein said assay comprises the steps of;

(i) isolating a population of cells from normal tissue of said individual,
and(ii) quantitatively determining the frequency of epimutation of a
particular gene in said population of cells, wherein the epimutation of
said gene is associated with said disease and said gene is other than one
that is subject to normal parent of origin-specific expression.

[0027]The determined epimutation frequency in the population of cells is
predictive of disease risk (i.e. predictive of a predisposition to said
disease) in said individual. For example, a positive risk of disease
(i.e. a predisposition to disease) may be predicted by a determined
epimutation frequency of at least 1 in 1×106 cells or, more
preferably, at least 1 in 1×103 cells or, most preferably, at
least 1 in 5×102 cells. Predictive frequencies of the
epimutation may vary according to the source of the cells assayed. That
is, the cells used in the assay may be from normal tissues such as, for
example, normal peripheral blood, normal hair follicles and normal tissue
from the buccal cavity, and determined frequencies which are predictive
of disease risk may vary across those different normal tissue types.

[0028]As used herein, the term "normal tissue" refers to any tissue which
is substantially healthy and not showing any significant symptoms or
signs of disease (e.g. the tissue is not cancerous) and includes all
normal somatic tissues. As indicated above, the cells used in the assay
may be from normal peripheral blood, normal hair follicles and normal
tissue from the buccal cavity. In addition to these, cells suitable for
assaying may be from other normal somatic tissues including normal
colonic mucosa. Preferably, the cells used in the assay are from normal
peripheral blood.

[0029]The assayed epimutation may be any of the well known epigenetic
modifications including DNA methylation (or other covalent modification
of DNA), histone and chromatin structure changes (e.g. histone
methylation, acetylation, phosphorylation or ubiquitination), or
association of other proteins in a complex with DNA of the affected locus
(e.g. HP1 and homologues).

[0030]The assayed epimutation is one which is associated with the disease
for which a predisposition is to be assessed. For example, the
epimutation is present in a chromosomal locus comprising a gene
implicated in the manifestation or development of a disease. Table 1
provides a list of genes implicated in over sixty diseases and the
epimutation may therefore be one present in a chromosomal locus
comprising at least one of the listed implicated genes, the assay thereby
being for the assessment of the disease associated with that gene(s). The
assayed epimutation may be present in the promoter of the gene(s) or
other regulatory region of the gene(s) and is associated with
transcriptional silencing of the gene(s).

[0031]Preferably, the assayed epimutation is one which is associated with
cancer. More preferably, the assayed epimutation is present in a tumour
suppressor gene such as hMLH1, hMSH2, APC 1A, APC 1B and p16.

[0032]Most preferably, the assayed epimutation is present in hMLH1.

[0033]The determination of the epimutation frequency in the assayed
population of cells may be achieved by either directly assaying
methylated cytosine (or other modification) on individual DNA strands or
by otherwise assaying pooled DNA/chromatin, without examining individual
DNA strands.

[0034]In order that the nature of the present invention may be more
clearly understood, preferred forms thereof will now be described with
reference to the following non-limiting example.

EXAMPLE 2

Materials and Methods

Patient Samples

[0035]188 individuals with a personal history of cancer from St Vincent's
Hospital (Sydney, NSW, Australia) and a further 50 individuals from the
Victorian Clinical Genetics Service (Melbourne, VIC, Australia) were
included in this study. Of these individuals, 65 were mutation-negative
following screening for deleterious germ-line changes in hMSH2, hMLH1 or
APC, while 18 had hyperplastic polyposis and the remaining 155 only had a
personal history of colorectal cancer.

[0036]DNA was extracted from peripheral blood, histologically-normal
colonic mucosa, buccal smears, hair follicles and sperm using a standard
phenol chloroform procedure (Sambrook et al 1989). To exclude the
possibility of contaminating somatic cells in the sperm, semen was sorted
on a FACSVantage DiVa (Becton Dickinson, Lexington, Ky., USA) prior to
DNA extraction. Sperm were identified on the basis of DNA content after
propidium iodide staining as described (Schoell et al 1999). Purity of
the sorted sperm was verified by FACS and microscopy.

MSI Analysis

[0037]Prior to the extraction of DNA from paraffin-embedded tissues, an
adjacent section was examined histologically to ensure that it contained
more than 60% tumour tissue. If this was not the case, foci of tumour
were microdissected. The microsatellite status of each tumour was
determined as previously described using the following primer sets: Bat
25, Bat 26, Bat 40, D5S346, D2S123, and D17S250 (Ward et al 2001).
Tumours with instability at two or more markers were considered
microsatellite unstable, while all others were designated as
microsatellite stable (MSS).

Immunohistochemical Staining for hMSH2 and hMLH1

[0038]Immunohistochemical analysis was performed in a DAKO autostainer on
dewaxed 4 μm paraffin sections (DAKO Corporation, Carpinteria, Calif.,
USA). Staining for hMLH1 and hMSH2 was as previously described, using
monoclonal anti-human hMLH1 antibody (1:200, Becton Dickinson, Lexington,
Ky., USA) and monoclonal anti-human hMSH2 antibody (1:400, Pharmingen,
San Diego, Calif., USA). Expression of hMLH1 or hMSH2 was considered to
be absent where there was no staining of tumour cells in the presence of
nuclear staining in nearby germinal follicle lymphocytes or in epithelial
cells in the base of adjacent non-neoplastic crypts. The immunostaining
analysis was reported without knowledge of results of MSI, germ-line or
CpG methylation results.

Methylation Screening Assays

[0039]Genomic DNA (2 μg) from each sample was then subject to bisulfite
modification (Frommer et al 1992). COBRA (combined bisulfite and
restriction analysis; Xiong and Laird 1997) was used as the screening
test for all genes. DNA was screened for epimutations in a panel of 10
candidate gene promoters, namely CDKN2A, hMLH1, HPP1, HIC1, RASSF1A,
BRCA1, APC 1A and 1B, Blm and O6MGMT PCR primers, reaction
conditions and post-PCR analysis information for the relevant assays are
shown in Table 2. A maximum of 100 ng of bisulfite treated DNA was used
in each reaction. In each PCR, positive and negative controls were
included, and these were the cell line RKO (gift from M Brattain) and the
peripheral blood DNA of a healthy donor, respectively.

Allelotyping of the hMLH1 Promoter

[0040]The single nucleotide polymorphism (SNP) at position -33 relative to
the hMLH1 transcription start site was determined by RFLP analysis of
unmodified genomic DNA. Primers, reaction conditions and restriction
digestion details are shown in Table 2.

Bisulfite Allelic Sequencing

[0041]Samples demonstrating a positive result on the COBRA screening assay
were examined in more detail using bisulfite sequencing. For hMLH1, this
involved a degenerate PCR designed to amplify both unmethylated and
methylated alleles, followed by cloning (pGEM-T, Promega) and sequencing
of individual alleles with the BigDyes system (ABI). The primers and PCR
conditions for amplification of this fragment are shown in Table 2. This
fragment was designed to include the G/A SNP described above.

[0042]Bisulfite-modified sperm DNA was analysed with a hybrid PCR
strategy, using a methylation-specific 5' primer and a
methylation-degenerate 3' primer, to enrich for methylated sequences
(Table 2).

Results and Discussion

[0043]Methylation of hMLH1 is Present in all Adult Tissue from Two
Individuals

[0044]Methylation at hMLH1 was detected in DNA from peripheral blood cells
of two of 94 individuals screened (FIG. 1A). The methylation extended
across the entire hMLH1 promoter, which is encompassed by regions named
A, B and C (FIG. 1B). Similar results were found when the A, B and C
screening assays were applied to DNA derived from these patients' hair
follicles, and buccal mucosa. These unrelated individuals, TT (male) and
VT (female) were aged 64 and 65 years, and both had a personal history of
multiple primary malignancies which had been successfully treated with
surgery alone. Clinical details of these individuals, as well as the
results of immunostaining for the mismatch repair proteins and
microsatellite testing of their tumours are shown in Table 3. All tumours
tested exhibited microsatellite instability (MSI), which is a hallmark of
mismatch repair deficiency. Loss of hMLH1 protein was confirmed in each
tumour by immunohistochemistry in the presence of normal staining for
hMSH2 (FIG. 2). While TT and VT had previously undergone germ-line
testing for mutations in the mismatch repair genes, no deleterious
mutations had been identified despite extensive screening with a number
of different methodologies.

[0045]To determine the distribution of CpG methylation within the tissues
of TT and VT, and construct detailed methylation maps, bisulfite allelic
sequencing was performed. By RFLP analysis, both TT and VT were found to
be heterozygous for a G/A single nucleotide polymorphism in hMLH1 at
position -33 relative to the transcription start site. Bisulfite
sequencing confirmed heterozygosity and also revealed that the
methylation in all tissues tested was restricted to the one parental
allele (FIG. 3). In both individuals, the affected allele was the G
genotype. The A allele was never found to be hypermethylated in any
tissue, although some CpG sites were occasionally methylated on some
alleles. The significance of this patchy methylation is unknown, but it
is not likely to be associated with hMLH1 silencing.

[0046]Mosaic methylation was observed in certain tissues from both
patients (FIG. 3). Patient VT was found to harbour some hypomethylated G
alleles in her peripheral blood (1/15), hair follicles (2/12) and buccal
mucosa (1/11). Of somatic tissues, patient TT exhibited mosaicism in hair
follicles only, with 1 out of 15 G alleles being hypomethylated.
Mosaicism was most evident in the sperm from TT. While the COBRA assay
yielded variably weak and negative results (data not shown), methylated
alleles were clearly present when the sperm were tested using an
MSP-COBRA hybrid PCR (FIG. 4). Of the alleles amplified, only G alleles
(6/16) showed hypermethylation. This is not an indication of frequency
however, because of the biased nature of the hybrid PCR. However, based
on the limit of sensitivity of the assays, it is estimated that between 1
in 500-1 in 1000 alleles may be methylated in the sperm of patient TT.

Methylation of hMLH1 in Colonic Mucosa of Individuals with Cancer

[0047]Normal colon tissue (n=133) from individuals with colorectal cancer
arising either sporadically or in the setting of hyperplastic polyposis,
was also screened. One individual with CpG methylation of hMLH1 in the
normal colon tissue was identified (FIG. 5A). This individual (NB1, a 65
yr old male), was assessed in more detail with bisulfite sequencing (FIG.
5B), and was found to be homozygous for the G SNP at -33 in hMLH1, thus
rendering the determination of the level of mosaicism impossible.
However, sequencing did confirm that NB1 harboured a significant
percentage of hypermethylated hMLH1 alleles in his normal colon (i.e.
14%; 3/21 alleles). It was also possible to assess the peripheral blood,
hair and buccal mucosa of NB1 for hMLH1 methylation by COBRA, although
all assays were negative. Patient NB1 had developed a renal cell cancer
at age 57 years and synchronous colorectal cancers in the caecum
(microsatellite unstable) and sigmoid colon (microsatellite stable) at
the age of 63 years in the setting of hyperplastic polyposis.

[0050]DNA was extracted from the peripheral blood as described in Example
1.

Methylation Screening Assays

[0051]Genomic DNA (2 μg) from each peripheral blood sample was
subjected to bisulfite modification as described in Example 1. The
bisulfite-treated DNA was then subjected to PCR with methylation-specific
primers (i.e. primers that hybridise with DNA in which the CpGs within
the primer binding sites are methylated) for the hMLH1 locus and the p16
locus (a tumour suppressor gene). Details of the primers used are listed
in Table 4.

Results and Discussion

[0052]Of the 22 healthy blood donors, 12 had some detectable level of
hypermethylated hMLH1 as demonstrated by the presence of a specific
product following PCR with methylation-specific primers. For the p16
gene, 18 of 29 healthy blood donors showed a detectable level of
hypermethylated alleles. For both genes, the PCR products were confirmed
as being hypermethylated by bisulfite sequencing. Representative
sequencing is shown in FIG. 6.

[0053]The results of this example indicate that healthy individuals
commonly carry a detectable level of cells in which the hMLH1 or p16 gene
is epimutated. Inactivation of one allele of either hMLH1 or p16, and
indeed many other tumour suppressor genes, is known to predispose a cell
to become malignant through loss or inactivation of the second allele. In
Example 1, two cancer patients (i.e. VT and TT) were described who carry
an epimutation in all or nearly all of their somatic cells; the normal
individuals studied in this example therefore presumably carry the
epimutation in only a small proportion of their somatic cells. It is,
however, considered that these cells are at risk of becoming malignant
through loss or inactivation of the second allele, and this risk is
higher than that of cells that do not carry the epimutation. Thus, it
follows that the more cells in an individual that carry the epimutation,
the higher will be that individual's risk of developing cancer; which is
analogous to disease caused by mosaic carriage of a genetic mutation.
Thus, the risk of developing cancer may be assessed by measuring the
proportion of somatic cells carrying a particular epimutation. Also, the
risk of developing other diseases that result from germline epimutation
(particularly, if the disease results when only one allele is
inactivated, i.e. haploinsufficiency, or when only a proportion of
somatic cells are affected by this loss, i.e. mosaicism) ought similarly
be assessed by measuring the proportion of somatic cells carrying a
particular epimutation.

[0054]Throughout this specification the word "comprise", or variations
such as "comprises" or "comprising", will be understood to imply the
inclusion of a stated element, integer or step, or group of elements,
integers or steps, but not the exclusion of any other element, integer or
step, or group of elements, integers or steps.

[0055]All publications mentioned in this specification are herein
incorporated by reference. Any discussion of documents, acts, materials,
devices, articles or the like which has been included in the present
specification is solely for the purpose of providing a context for the
present invention. It is not to be taken as an admission that any or all
of these matters form part of the prior art base or were common general
knowledge in the field relevant to the present invention as it existed in
Australia or elsewhere before the priority date of each claim of this
application.

[0056]It will be appreciated by persons skilled in the art that numerous
variations and/or modifications may be made to the invention as shown in
the specific embodiments without departing from the spirit or scope of
the invention as broadly described. The present embodiments are,
therefore, to be considered in all respects as illustrative and not
restrictive.